Extracellular vesicles (EVs) are heterogenous membrane-bound vesicles released from various origins. EVs play a crucial role in cellular communication and mediate several physiological and pathological processes, highlighting their potential therapeutic and diagnostic applications. Due to the rapid increase in interests and needs to elucidate EV properties and functions, numerous isolation and separation approaches for EVs have been developed to overcome limitations of conventional techniques, such as ultracentrifugation. This review focuses on recently emerging and modern EV isolation and separation techniques, including size-, charge-, and affinity-based techniques while excluding ultracentrifugation and precipitation-based techniques due to their multiple limitations. The advantages and drawbacks of each technique are discussed together with insights into their applications. Emerging approaches all share similar features in terms of being time-effective, easy-to-operate, and capable of providing EVs with suitable and desirable purity and integrity for applications of interest. Combination and hyphenation of techniques have been used for EV isolation and separation to yield EVs with the best quality. The most recent development using an automated on-line system including selective affinity-based trapping unit and asymmetrical flow field-flow fractionation allows reliable isolation and fractionation of EV subpopulations from human plasma.

An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation (IAC - AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61- positive EVs and CD9- positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g. exomeres and exosomes) for further analysis. Field-emission scanning electron microscope elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in ng/mL range using hydrophilic interaction liquid chromatography – tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37-80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84-6.6%.

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.

Raman spectroscopy together with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS) was employed to characterize exomere- (<50 nm) and exosome-sized (50-80 nm) EVs isolated from human plasma by the novel on-line immunoaffinity chromatography - asymmetric flow field-flow fractionation method. CD9⁺, CD63⁺, and CD81⁺ EVs were selected to represent general EV subpopulations secreted into plasma, while CD61⁺ EVs represented the specific EV subset derived from platelets. Raman spectroscopy could distinguish EVs from non-EV particles, including apolipoprotein B-100-containing lipoproteins, signifying its potential in EV purity assessment. Moreover, platelet-derived (CD61⁺) EVs of both exomere and exosome sizes were discriminated from other EV subpopulations due to different biochemical compositions. Further investigations demonstrated composition differences between exomere- and exosome-sized EVs, confirming the applicability of Raman spectroscopy in distinguishing EVs, not only from different origins but also sizes. In addition, fatty acids that act as building blocks for lipids and membranes in EVs were studied by GCxGC-TOF-MS. The results achieved highlighted differences in EV fatty acid compositions in both esterified (membrane lipids) and non-esterified (free fatty acids) fractions, indicating possible differences in membrane structures, biological functions, and roles in cell-to-cell communications of EV subpopulations.